CN107202890A - Fast diagnosis reagent and the preparation containing the kit of the reagent and the kit and detection method for brain injury - Google Patents
Fast diagnosis reagent and the preparation containing the kit of the reagent and the kit and detection method for brain injury Download PDFInfo
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- CN107202890A CN107202890A CN201710396942.XA CN201710396942A CN107202890A CN 107202890 A CN107202890 A CN 107202890A CN 201710396942 A CN201710396942 A CN 201710396942A CN 107202890 A CN107202890 A CN 107202890A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
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Abstract
The invention belongs to immunoassay technology field, more particularly to a kind of fast diagnosis reagent and the preparation containing the kit of the reagent and the kit and detection method for brain injury.The reagent and kit include S100, GFAP, UCHL1 obtained monoclonal capture antibody respectively；The kit also includes detection antibody, the obtained polyclonal antibody of S100, GFAP, UCHL1 difference.Beneficial effects of the present invention are as follows：Brain injury can comprehensively be detected, the physiology after brain damage and structure change can be preferably judged, preferably centering Mild brain injury patient carries out antidiastole, with sensitivity high accuracy it is strong the characteristics of, its sensitivity and accuracy reach more than 96%.
Description
Technical field
The invention belongs to immunoassay technology field, more particularly to a kind of quick diagnosis for brain injury is tried
Agent and the preparation containing the kit of the reagent and the kit and detection method.
Background technology
Craniocerebral trauma (TBI) has been to cause the whole world to be died of illness and invalid major public health problem at present.The U.S. has every year
235 × 103 non-lethal Patients with Cerebral Trauma hospitalizations, 43.30% leaves different degrees of deformity.China's craniocerebral trauma year
Incidence is to have 783 in every 100,000 people, and wherein Glasgow stupor scales (GCS), which score, forgets the time after≤8 points and wound
(PTA)>24 h severe traumatic head injury accounts for 10%.
The children and adult for having more than 1,500,000 every year in the U.S. suffered from have thousands of army in cerebral concussion, global range
People suffer from slight traumatic brain injury.Current detection can't determine the degree damaged or whether 15-30% injury
Patient can be subjected to serious, permanent cognitive defect, change and balance between such as processing speed, working memory and a variety of thoughts
Ability.Current diagnosis according to mainly clinical symptoms and neuropsychological test, iconography and biochemistry detection as supplementary means,
Available for excluding amnesia caused by other causes of disease.When clinical symptoms performance is obvious, existing medical procedure goes to reverse
Through becoming extremely difficult.So prophylactic to occur, early diagnosis becomes particularly important.Present research finds brain injury
Pathology lesion can not actually be detected with traditional image, biochemical apparatus.One new biomarker can be accurate
There is permanent cognitive function after white matter fiber tract structural damage and slight cerebral trauma in prediction, brain injury patients sustainable development
Obstacle.
Brain damage mark refers to during brain tissue and cell genesis and development, is synthesized, released in itself by brain cell
Put, or the class marker substances by reacting brain injury and producing.These materials are not present in normal adult either to exist
The level occurred in cancer patient is significantly higher than normal person.The innovation and creation of Application No. 201580018413.5 disclose one kind
Traumatic brain injury and nervus retrogression biomarker, method and system.This method includes following in detection Patient Sample A
It is one or more：Ubiquitin c-terminal hydrolase-l 1 (UCH L1), GFAP (GFAP), the family member of aldehyde dehydrogenase 1
Chain (NFM) or neurofilament light chain (NFL), α cynapse core eggs in L1 (ALDH1L1), phosphorylated neurofilament heavy chain (pNFH), neurofilament
In vain, visinin sample albumen 1 (VILIP 1) and S100B.But, with the practical experience of present inventor, due to individual difference
Presence, the clinical test of the above-mentioned single factor or two factors is all failure, and there is also larger for the combination of multiple factors
It is uncertain.Above-mentioned patent is only a layout patent, and not yet presence can exclude the detection side of individual difference in the prior art
Method.
The content of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of quick diagnosis for brain injury
Reagent and the preparation containing the kit of the reagent and the kit and detection method, reach on the premise of individual difference is excluded
The purpose of brain injury can quickly be made a definite diagnosis.
To achieve these goals, technical scheme is as follows：For the fast diagnosis reagent of brain injury,
Including capture antibody, it is characterised in that：The capture antibody includes S100, GFAP, UCHL1 obtained monoclonal antibody respectively.
The diagnostic reagent also includes detection antibody, and it is obtained many respectively that the detection antibody includes S100, GFAP, UCHL1
Clonal antibody.
As an improvement, the capture antibody is prepared by following steps：(1) preparation of antigen：By S100, GFAP, UCHL1 base
The expression of albumen is realized because being cloned into carrier for expression of eukaryon, and in mammalian cell, required antigen is obtained after purification, should
Antigen is used as standard items；(2) preparation of antibody is captured：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal to resist
Body, the monoclonal antibody as this reagent capture antibody.
As an improvement, the detection antibody is prepared by following steps：(1) preparation of antigen：S100, GFAP, UCHL1
Gene cloning realizes in mammalian cell the expression of albumen to carrier for expression of eukaryon, and antigen is obtained after purification；(2) examine
Survey the preparation of antibody：By above-mentioned antigen immune mammal, it is detection antibody to obtain corresponding polyclonal antibody.
The concentration reference point of S100, GFAP, UCHL1 in serum is respectively 1.2,1.5 and 0.6ng/ml, if having 2 in 3
~3 are then diagnosed as brain injury more than concentration reference point.
Kit comprising above-mentioned diagnostic reagent includes following preparation process：(1) preparation of antigen：By S100, GFAP,
UCHL1 gene clonings realize in mammalian cell the expression of albumen to carrier for expression of eukaryon, needed for obtaining after purification
Antigen；(2) preparation of antibody is captured：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the monoclonal
Antibody as this kit capture antibody；(3) preparation of antibody is detected：Above-mentioned antigen immune mammal is obtained accordingly
Polyclonal antibody, the polyclonal antibody as this kit detection antibody；(4) capture antibody coating：1. with carbonate/
Concentration is coated with the hole of microtiter plate by bicarbonate buffer for 1 μ g/ml capture antibody；2. covers microtiter plate simultaneously
It is incubated overnight at 4 DEG C；3. discards coating buffer, and washs microtiter plate twice with cleaning solution, is added every time in micropore
200 μ l PBST, remove cleaning solution and remaining drop, dry and be put in 4 DEG C of environment；(5) close：200 μ l closings are added per hole
Remaining protein binding site in buffer solution, closing coating hole.Capture antibody is coated in the hole of microtiter plate in advance.
Kit comprising above-mentioned diagnostic reagent includes following preparation process：Including following detecting step：(1) system of antigen
It is standby：By S100, GFAP, UCHL1 gene cloning to carrier for expression of eukaryon, and realize in mammalian cell the expression of albumen；
(2) preparation of antibody is captured：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the monoclonal antibody is made
For the capture antibody of this kit；(3) preparation of antibody is detected：Above-mentioned antigen immune mammal is obtained corresponding polyclonal
Antibody, the polyclonal antibody as this kit detection antibody；(4) capture antibody coating：1. carbonate/bicarbonates
Concentration is coated with the hole of microtiter plate by salt buffer for 1 μ g/ml capture antibody；2. covers microtiter plate and at 4 DEG C
It is incubated overnight；3. discards coating buffer, and washs microtiter plate twice with cleaning solution, adds 200 μ l in micropore every time
PBST, removes cleaning solution and remaining drop, dries and be put in 4 DEG C of environment；(5) close：1. is in each hole of microtiter plate
Add remaining protein binding site in 200 μ l Block buffers, closing coating hole；2. covers microtiter plate and 37
DEG C be incubated 1 hour；(6) it is loaded：1. 100 μ l samples are added to each hole by, are incubated 60 minutes at 37 DEG C；2. discards sample,
And wash microtiter plate three times, 200 μ l PBST are added in micropore every time；3. by 100 μ l concentration be 0.5 μ g/ml inspection
Survey antibody and be added to each hole；4. covers microtiter plate and is incubated 1 hour at 37 DEG C；5. washs microtiter plate with PBST
Four times；6. adds 100 μ l mark secondary antibodies；7. covers microtiter plate and is incubated 1 hour at 37 DEG C；8. washs micro- with PBST
Measure titer plate four times；(7) detect：1. TMB solution is added to each hole by, is incubated 15-30 minutes, adds isometric termination
Liquid, then reads optical density at 450nm；(8) judge：The concentration reference point of S100, GFAP, UCHL1 in serum is respectively
1.2,1.5 and 0.6ng/ml, is diagnosed as brain injury in 3 if having 2~3 to exceed concentration reference point.
The present invention filters out 3 brain damage label S100, GFAP and UCHL1 groups from numerous brain damage labels
Into quick diagnosis reagent kit.Whether it is light with regard to that can make a definite diagnosis as long as the testing result for having 2 factors in three factors has reached threshold values
Moderate traumatic brain injury.The kit overcomes individual difference, with the characteristics of sensitivity is high, accuracy is strong, its sensitivity and accurate
Property reaches more than 96%.
Brief description of the drawings
Fig. 1 is used for the schematic diagram of mild or moderate brain injury patients quick diagnosis reagent kit for the present invention；
Fig. 2 detects for the present invention for mild or moderate brain injury patients quick diagnosis reagent kit in normal human blood
S100/GFAP/UCHL1 concentration.
Embodiment
Embodiment 1
For mild or moderate brain injury patients fast diagnosis reagent and kit, including capture antibody, capture antibody, close slow
Fliud flushing, standard items, labelled antibody, cleaning solution and nitrite ion etc..The labelled antibody marks secondary antibody from HRP.The capture is anti-
Body includes S100, GFAP and UCHL1 obtained monoclonal antibody respectively；Pass through the common quick inspection to S100, GFAP and UCHL1
Survey, Detection accuracy and sensitivity can be effectively improved, effectively overcome only according to not enough caused by the single factor of detection.Fig. 1
It is used for the schematic diagram of mild or moderate brain injury patients quick diagnosis reagent kit for the present invention.
The capture antibody is that S100, GFAP, UCHL1 are cloned into carrier for expression of eukaryon, and in mammalian cell
The expression of albumen is realized, corresponding antigen is obtained after purification, the corresponding monoclonal that the antigen immune mammal is obtained resists
Body.
The step of preparation method of the capture antibody includes is as follows：(1) preparation of antigen：Pass through the method for molecular cloning
S100, GFAP, UCHL1 gene cloning to carrier for expression of eukaryon, and realize in mammalian cell the expression of albumen, purifying
After obtain antigen, this antigen can also be used as standard items.The preparation of the antigen can also use existing conventional method to prepare,
This is no longer burdensome；(2) preparation of antibody is captured：By above-mentioned antigen immune mammal, obtain corresponding monoclonal antibody to catch
Obtain antibody.The mammal is mouse and rabbit, preferably mouse.The preparation of the capture antibody can also use existing routine
Prepared by method, no longer burdensome herein.
The detection antibody includes S100, GFAP, UCHL1 obtained polyclonal antibody respectively.
The detection antibody is that S100, GFAP, UCHL1 are cloned into carrier for expression of eukaryon, and in mammalian cell
The expression of albumen is realized, corresponding antigen is obtained after purification, the corresponding Anti-TNF-α that the antigen immune mammal is obtained
Body.The step of preparation method of the detection antibody includes is as follows：(1) preparation of antigen：Pass through the method handle of molecular cloning
S100, GFAP, UCHL1 gene cloning realize in mammalian cell the expression of albumen to carrier for expression of eukaryon, after purification
Antigen is obtained, this antigen can also be used as standard items；(2) preparation of antibody is detected：By above-mentioned antigen immune mammal, obtain
Corresponding polyclonal antibody is detection antibody.The mammal is mouse and rabbit, preferably rabbit.
Wherein, the capture antibody can be coated in the hole of the microtiter plate of PVC materials in advance, can simplify step
Suddenly, detection efficiency is improved.
For the preparation method of mild or moderate brain injury patients quick diagnosis reagent kit, it comprises the following steps：
(1) preparation of antigen：S100, GFAP, UCHL1 gene cloning to eukaryotic expression are carried by the method for molecular cloning
Body, and the expression of albumen is realized in mammalian cell, required antigen is obtained after purification, wherein, the antigen can also be made
Used for standard items；
(2) preparation of antibody is captured：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the Dan Ke
Grand antibody as this kit capture antibody；
(3) preparation of antibody is detected：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, described many grams
Grand antibody as this kit detection antibody；
(4) capture antibody coating：
1. concentration is coated in micro drop by with carbonate/bicarbonate buffer solution (pH9.6) for l μ g/ml capture antibody
In the hole of fixed board；
2. covers microtiter plate with bond plastic product and is incubated overnight at 4 DEG C；
3. discards coating buffer (the capture antibody of carbonate/bicarbonate buffer solution dilution), and washs micro with cleaning solution
Titer plate adds 200 μ 1PBST (phosphate Tween buffer) in micropore twice, every time, and gently whipping is micro above tank
Titer plate, removes cleaning solution, pats microtiter plate on paper handkerchief, removes remaining drop, dry be put in it is standby in 4 DEG C of environment
With；The cleaning solution is a certain amount of Tween20 of addition, the mass percent of the Tween20 in PBS (phosphate buffer)
Concentration is 0.05%；
(5) close
1. (is containing 1.2%BSA's (bovine serum albumin(BSA)) in every hole addition 200 μ 1 Block buffers of microtiter plate
PBS (phosphate buffer)), for closing remaining protein binding site in coating hole；
2. covers microtiter plate with bond plastic product and is incubated at least 1 hour at 37 DEG C, it is preferred that be incubated
Night.
For the detection method of mild or moderate brain injury patients quick diagnosis reagent kit, it comprises the following steps：
(1) preparation of antigen：S100, GFAP, UCHL1 gene cloning to eukaryotic expression are carried by the method for molecular cloning
Body, and the expression of albumen is realized in mammalian cell, required antigen is obtained after purification, wherein, the antigen can also be made
Used for standard items；
(2) preparation of antibody is captured：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the Dan Ke
Grand antibody as this kit capture antibody；
(3) preparation of antibody is detected：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, described many grams
Grand antibody as this kit detection antibody；
(4) capture antibody coating：
1. concentration is coated with microtitration by carbonate/bicarbonates buffer solution (pH9.6) for l μ g/ml capture antibody
The hole of plate；
2. covers microtiter plate with bond plastic product and is incubated overnight at 4 DEG C；
3. discards coating buffer (the capture antibody of carbonate/bicarbonate buffer solution dilution), and washs micro with cleaning solution
Titer plate adds 200 μ 1PBST in micropore twice, every time, and (phosphate Tween buffer can contain 0.05% Tween-20
PH value is 7.4 phosphate buffer), the gently whipping microtiter plate above tank removes cleaning solution, patted on paper handkerchief
Microtiter plate, removes remaining drop, dry be put in it is standby in 4 DEG C of environment；The cleaning solution is PBS (phosphate buffer)
Middle to add a certain amount of Tween 20, Tween 20 mass percent concentration is 0.05%；
(5) close
1. is added in each hole of microtiter plate in 200 μ l Block buffers (1.2%BSA/PBS), closing coating hole
Remaining protein binding site；
2. covers microtiter plate with bond plastic product and is incubated at least 1 hour at 37 DEG C, it is preferred that at 4 DEG C
It is incubated overnight；
(6) it is loaded
1. the 100 μ Ι samples for suitably diluting (20 times of dilution) are added to each hole by, are incubated 60 minutes at 37 DEG C；Will
Obtain accurate quantitative result, it is common practice that compare the signal of unknown sample and standard curve.Each ELISA Plate must be surveyed
Standard items (double measure or triplicate) and blank sample are determined, to ensure accuracy；
2. discards sample, and washs microtiter plate three times, adds 200 μ 1PBST (phosphate tweens in micropore every time
Buffer solution)；
3. 100 μ Ι concentration are added to each hole of microtiter plate by for 0.5 μ g/ml detection antibody；
4. covers microtiter plate with bond plastic product and is incubated 1 hour at 37 DEG C；
5. washs microtiter plate with PBST four times；
6. adds 100 μ Ι mark secondary antibodies, and it is just diluting 10000 times (1 in roS before:10000).The mark
Remember that secondary antibody can mark goat-anti rabbit for HRP.
7. covers microtiter plate with bond plastic product and is incubated 1 hour at 37 DEG C.
8. washs microtiter plate with PBST four times.
(7) detect
1. TMB (3,3', 5,5 tetramethyl benzidine) solution is added to each hole by, is incubated 15-30 minutes, the body such as addition
Long-pending terminate liquid (2M H2S04), then reads optical density at 450nm.
2. the data that is obtained by serial dilutions draw standard curve, and concentration is marked in X-axis (logarithmic scale), and extinction
Scale is in Y-axis (lineal scale).Sample concentration is drawn on this standard curve by interpolation method.
(8) judge
The concentration reference point of S100, GFAP, UCHL1 in serum is respectively 1.2,1.5 and 0.6ng/ml, if having 2 in 3
~3 are then diagnosed as brain injury more than concentration reference point.
The present invention is used for mild or moderate brain injury patients quick diagnosis reagent kit to be had including tri- indexs of S100, GFAP, UCHL1
Two rises simultaneously can be used for clinically by 3 brain damage marks in detection blood as the standard of diagnosis the severity of brain injury
The extent of injury of the height alignment degree brain injury patients of thing level carries out dynamic evaluation.It can be additionally used for clinically damaging brain
Hinder the application of patient's later stage symptom judgement.
To determine that can this kit be used to carry out dynamic evaluation to the therapeutic effect of oophoroma, we have collected in 8 parts
The serum of patients with mild head injury (GCS 9-12).The testing result of 8 patients is as shown in the table, as a result shows brain injury patients
There were significant differences with the level of S100, GFAP, UCHL1 in the serum of normal person's (3 parts).S100, GFAP in patients serum,
UCHL1 level can rise significantly, point out this kit to be used for the carry out dynamic evaluation to brain injury patients.
Fig. 2 detects for the present invention for mild or moderate brain injury patients quick diagnosis reagent kit in normal human blood
S100/GFAP/UCHL1 concentration, present invention determine that the reference concentration of S100, GFAP, UCHL1 in serum is respectively 1.2,
1.5 and 0.6ng/ml, three have two to exceed reference point as the standard of brain injury.
Preferred embodiment of the invention described in detail above, it will be appreciated that the ordinary skill of this area is without wound
The property made work just can make many modifications and variations according to the design of the present invention.Therefore, all technical staff in the art
According to present inventive concept in prior art basis by logic analysis, reasoning or according to the limited available technology of experiment
Scheme, should be among the protection domain determined by the claims.
Claims (9)
1. for the fast diagnosis reagent of brain injury, including capture antibody, it is characterised in that：The capture antibody includes
The obtained monoclonal antibody of S100, GFAP, UCHL1 difference.
2. it is used for the fast diagnosis reagent of brain injury as claimed in claim 1, it is characterised in that：Also include detection anti-
Body, the detection antibody includes S100, GFAP, UCHL1 obtained polyclonal antibody respectively.
3. it is used for the fast diagnosis reagent of brain injury as claimed in claim 1, it is characterised in that：The capture antibody
Prepared by following steps：(1) preparation of antigen：By S100, GFAP, UCHL1 gene cloning to carrier for expression of eukaryon, and in lactation
The expression of albumen is realized in zooblast, required antigen is obtained after purification, the antigen is used as standard items；(2) antibody is captured
Preparation：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal antibody, the monoclonal antibody is used as this reagent
Capture antibody.
4. it is used for the fast diagnosis reagent of brain injury as claimed in claim 2, it is characterised in that：The detection antibody
Prepared by following steps：(1) preparation of antigen：S100, GFAP, UCHL1 gene cloning to carrier for expression of eukaryon, and feeding
The expression of albumen is realized in newborn zooblast, antigen is obtained after purification；(2) preparation of antibody is detected：Above-mentioned antigen immune is fed
Newborn animal, it is detection antibody to obtain corresponding polyclonal antibody.
5. it is used for the fast diagnosis reagent of brain injury as claimed in claim 1, it is characterised in that：S100、GFAP、
Concentration reference points of the UCHL1 in serum is respectively 1.2,1.5 and 0.6ng/ml, if there is 2~3 to exceed concentration reference in 3
Value is then diagnosed as brain injury.
6. the quick diagnosis reagent kit for brain injury, it is characterised in that：It is any including such as above-mentioned Claims 1 to 4
Diagnostic reagent described in.
7. it is used for the quick diagnosis reagent kit of brain injury as claimed in claim 6, it is characterised in that：The capture is anti-
Body is coated in the hole of microtiter plate in advance.
8. it is used for the quick diagnosis reagent kit of brain injury as claimed in claim 7, it is characterised in that：Including following system
Standby step：(1) preparation of antigen：By S100, GFAP, UCHL1 gene cloning to carrier for expression of eukaryon, and in mammalian cell
The middle expression for realizing albumen, obtains required antigen after purification；(2) preparation of antibody is captured：Above-mentioned antigen immune lactation is moved
Thing obtains corresponding monoclonal antibody, the monoclonal antibody as this kit capture antibody；(3) system of antibody is detected
It is standby：Above-mentioned antigen immune mammal is obtained into corresponding polyclonal antibody, the polyclonal antibody as this kit inspection
Survey antibody；(4) capture antibody coating：1. concentration is coated with by carbonate/bicarbonates buffer solution for 1 μ g/ml capture antibody
The hole of microtiter plate；2. covers microtiter plate and is incubated overnight at 4 DEG C；3. discards coating buffer, and is washed with cleaning solution
Wash microtiter plate twice, add 200 μ l PBST in micropore every time, remove cleaning solution and remaining drop, dry and be put in 4
In DEG C environment；(5) close：Remaining protein binding site in 200 μ l Block buffers, closing coating hole is added per hole.
9. it is used for the quick diagnosis reagent kit of brain injury as claimed in claim 7, it is characterised in that：Including following inspection
Survey step：(1) preparation of antigen：By S100, GFAP, UCHL1 gene cloning to carrier for expression of eukaryon, and in mammalian cell
The middle expression for realizing albumen；(2) preparation of antibody is captured：Above-mentioned antigen immune mammal is obtained into corresponding monoclonal to resist
Body, the monoclonal antibody as this kit capture antibody；(3) preparation of antibody is detected：By above-mentioned antigen immune lactation
Animal obtains corresponding polyclonal antibody, the polyclonal antibody as this kit detection antibody；(4) antibody bag is captured
Quilt：1. concentration is coated with the hole of microtiter plate by carbonate/bicarbonates buffer solution for 1 μ g/ml capture antibody；2. is sealed
Lid microtiter plate is simultaneously incubated overnight at 4 DEG C；3. discards coating buffer, and washs microtiter plate twice with cleaning solution, every time
200 μ l PBST are added in micropore, cleaning solution and remaining drop is removed, dries and be put in 4 DEG C of environment；(5) close：1. exists
Add remaining protein binding site in 200 μ l Block buffers, closing coating hole in each hole of microtiter plate；2. is sealed
Lid microtiter plate is simultaneously incubated 1 hour at 37 DEG C；(6) it is loaded：1. 100 μ l samples are added to each hole by, and 60 are incubated at 37 DEG C
Minute；2. discards sample, and washs microtiter plate three times, adds 200 μ l PBST in micropore every time；3. is dense by 100 μ l
The detection antibody spent for 0.5 μ g/ml is added to each hole；4. covers microtiter plate and is incubated 1 hour at 37 DEG C；5. is used
PBST washings microtiter plate four times；6. adds 100 μ l mark secondary antibodies；7. covers microtiter plate and small in 37 DEG C of incubations 1
When；8. washs microtiter plate with PBST four times；(7) detect：1. TMB solution is added to each hole by, is incubated 15-30 points
Clock, adds isometric terminate liquid, and optical density is then read at 450nm；(8) judge：S100, GFAP, UCHL1 are in serum
Concentration reference point be respectively in 1.2,1.5 and 0.6ng/ml, 3 if be diagnosed as if thering are 2~3 to exceed concentration reference point it is light in
Spend brain damage.
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| CN201710396942.XA CN107202890A (en) | 2017-05-31 | 2017-05-31 | Fast diagnosis reagent and the preparation containing the kit of the reagent and the kit and detection method for brain injury |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111094983A (en) * | 2017-12-09 | 2020-05-01 | 雅培实验室 | Methods of using Glial Fibrillary Acidic Protein (GFAP) and/or ubiquitin carboxy-terminal hydrolase L1(UCH-L1) to aid in the diagnosis and evaluation of patients who have suffered orthopedic injury and who have suffered or may have suffered a head injury such as mild Traumatic Brain Injury (TBI) |
| CN114624448A (en) * | 2022-03-18 | 2022-06-14 | 北京美联泰科生物技术有限公司 | Kit for detecting acidic protein in glial fibers |
| CN115097138A (en) * | 2022-05-26 | 2022-09-23 | 北京美联泰科生物技术有限公司 | Application of buffer solution in PGP9.5 detection kit |
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| CN111094983A (en) * | 2017-12-09 | 2020-05-01 | 雅培实验室 | Methods of using Glial Fibrillary Acidic Protein (GFAP) and/or ubiquitin carboxy-terminal hydrolase L1(UCH-L1) to aid in the diagnosis and evaluation of patients who have suffered orthopedic injury and who have suffered or may have suffered a head injury such as mild Traumatic Brain Injury (TBI) |
| CN114624448A (en) * | 2022-03-18 | 2022-06-14 | 北京美联泰科生物技术有限公司 | Kit for detecting acidic protein in glial fibers |
| CN115097138A (en) * | 2022-05-26 | 2022-09-23 | 北京美联泰科生物技术有限公司 | Application of buffer solution in PGP9.5 detection kit |
| CN115097138B (en) * | 2022-05-26 | 2023-11-17 | 北京美联泰科生物技术有限公司 | Application of buffer solution in PGP9.5 detection kit |
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